Project Summary/Abstract Tumor heterogeneity and clonal evolution play key roles in cancer progression and the response to therapy. However, clonal evolution has been difficult to study in solid tumors such as bladder cancer, in part due to the lack of amenable model systems. In this Project, we propose to use patient-derived bladder tumor organoids as a new model system to pursue longitudinal studies of tumor evolution and its role in drug response and resistance. In preliminary studies, we have developed culture conditions for the establishment of patient-derived organoid lines from bladder cancer tissues that range from papillary non-invasive tumors to muscle-invasive cancer, and have shown that these organoid lines recapitulate the histopathological and molecular features of their corresponding parental tumors. In particular, we have found that these organoid lines display changes in mutational profiles during serial passaging in culture that are consistent with clonal evolution. We have further demonstrated that these lines can be used to assay drug response, and that these responses can be validated in orthotopic xenografts from these organoids in vivo. We will now use these patient-derived bladder cancer organoid models to investigate clonal evolution and drug response in bladder cancer by pursuing three specific aims: 1) Analysis of heterogeneity and tumor evolution in bladder cancer organoids by examining the maintenance of heterogeneity during serial passaging and by investigating how genome instability and epigenetic dysregulation drive clonal evolution; 2) Investigation of chemotherapy response in bladder cancer organoids by analyzing the role of the nucleotide excision repair pathway gene ERCC2 in differential response to cisplatin treatment; and 3) Analysis of clonal evolution in drug resistance of bladder cancer organoids using lentiviral-mediated barcoding and single-cell RNA sequencing to perform longitudinal analyses of clonal tumor populations during the emergence of drug resistance in culture. These studies will be greatly facilitated by the biobank of patient-derived organoid lines and xenografts that will be generated by the Bladder Cancer Models Core, histopathology and targeted exome sequencing performed by the Molecular Pathology Core, and bioinformatic and biostatistical support from the Administrative Core. Our work will also be highly integrated with Projects 1 and 2 through our analyses of the potential roles of the epigenetic regulators KDM6A and ARID1A in driving tumor heterogeneity and evolution, while at the same time, our studies of patient-derived bladder tumor organoids will provide valuable reagents and insights for the other Projects.